Process of extracting physiologically active materials from animal tissues



Patented May 9, 1939 2,353,233 PEQQESS fill EXTRACWG rnirsroaoerw GALE?!AC'EHVE TIESWES Fri-@Itll Hi'avard L. lieil, Chicago, m, Mignon toArmour and Company, lillcdcago,

llllllnois ml... a corporation of No Application some t, ices,

Serial No. 533

6 Claims.

This invention relates to processes oi isolating physiologically activematerials, such as hormones and enzymes, from animal tissue, and itcomprises processes wherein the animal tissue is finely minced and thehydrogen ion concentration thereof then so changed that the hormones orenzymes contained therein are released, become associated with thenatural aqueous juice Of the tissue, and arerecovered therefrom.

Many different kinds of hormones and enzymes are present in animaltissues, for example hog liver contains physiologically active materialsuseful in the treatment of certain body conditions. Epinephrin can beobtained from the adrenals, thyroxlne from the thyroids, insulin fromthe pancreas, secretin from the duodenum, etc. Present methods ofextracting these hormones depend upon the action of acidified solutions,usually alcoholic, which physically extract the hormone. In other words,present extraction methods, as the word implies, maize use of solventsfor either the hormone itself, or the hormone which has been activatedby theacid in the extracting liquid.

For example, secretin can be extracted from the duodenum byprocessesinvolving the treatment of minced duodenum with relativelylarge volumes of acidified alcohol solutions and the secretinsubsequently recovered from its extraction liquid. Many extractionprocesses now used require a heating step either in the extraction perse or in the purification of the extracted hormone. Others require theuse of precipitating agents to free the extract of impurities and stillothers must utilize alcoholic solutions strong enough to. precipitateproteins. All of these methods have disadvantages. Many of thephysiologically active materials it is desired to isolate are destroyedby heating, still others are precipitated and lost by the ammoniumsulfate used as a protein precipitant, and still others are not solublein the strong alcohol solutions required to free the material ofproteins. In the latter instance, large quantities of the desiredhormone are actually precipitated with the protein impurities. Moreover,the. wide use of alcoholic extraction solutions is expensive since isnot complete.

01 late, methods using absorbent materials have been proposed. This hasbeen more an effort to avoid the addition of large amounts of aqueousextracting solutions. Although solid absorbent materials are animportant step forward in the art, such absorbent action is usuallyrather specific and is not particularly satisfactory. The

alcohol recovery hormone must be recovered from the absorbent and indoing this, many of the disadvantages sought to be overcome by theabsorbent method actually reappear.

What is desired in this art is some method of isolating hormones andother physiologically active constituents of animal tissue which avoidsthe use of large volumes of extraction liquids. At the same time, such amethod should avoid the necessity for drastic purification whichinvariably results in marked losses of the very hormone it is desired toisolate. In other words, the art has long desired a method by means ofwhich the hormonecan be isolated under conditions which avoid thenecessity for drastic purification methods and large volumes ofextraction solutions.

I have now discovered a method which meets the above requirements. Mymethod can be characterized as a solventless extraction-method in that Ido not treat the animal tissue with a solvent or an extraction fluid. Inmy method, I am able to use the normal tissue juice as the liquid phasein which the physiologically active materials collect. In most glandularmaterial the natural tissue juice is roughly 70 to 80 percent of thetissue. In my process I so treat the glandular material that thehormones are liberated from the cells of the tissue and collect ordissolve in the tissue juice. Consequently, I am able to avoid treatmentof the tissue with large volumes of solvents and thus I avoid much ofthe expense hitherto considered an evil in the extraction of animaltissue.

Before proceeding with a detailed description oi the application of myinvention to certain specific materials, it will aid in anunderstandingof the principles of my invention ii I describe thecolloidal chemical phenomena which I put to use. This will likewise helpto distinguish the fundamental principles of my invention fromextraction methods hitherto used in the art.

Hormones and similar physiologically active materials are normallyintracellular. That is to say, they are found inside the individualcells 01 the animal tissue. When animal tissue, such as the duodenum,1's finely minced there is obtained a pulpy mass composed of arelatively large volume of tissue juice in which the individual cells ofthe tissue can be visualized as in suspension. In other words, theminced material can be looked upon as an aqueous pulp of animal tissuecells. Naturally the cells are of microscopic size. The hormonewhich itis desired to isolate is within the cells themselves and is notassociated present processes with the continuous liquid phase of thepulp. In the action of large volumes of aqueous or alcoholic acidsolutions appears to break down the cells chemically and thus liberatethe hormone. In some instances, however, the action of large volumes ofacidified water or alcohol may not result in an actual splitting orbreak down or the protein cell. Some of the hormones may be looselylinked chemically to the cell by residual valence bonds and the smallamount of acid in the extractingsolution may destroy this valence bondand free the hormone from the cell. Just what the true explanation iscannot be stated with finality. The fact remains, however, that theusual methods of separating the hormone from the protein cells withwhich it is associated tend to destroy some of the hormone. All methodsinvolving heat, the addition oi! large volumes of alcohol, or saltingout inevitably lead to losses oi hormone which could otherwise berecovered.

The present invention is based upon the application of colloidalchemical principles not hitherto used in this art. In my process I causethe cells of the tissue to swell until finally a point is.reached wherethe cell wall ruptures and the hormone is released. The, aqueous fluidused for swelling the protein cell is the natural tissue juice althoughI do add small quantities of water as a carrier for the agents whichcause the cell to swell. The amount of water added, however, is farbelow that customarily used as a solvent or extraction medium for thehormone.

To put it another way, the hormone can be considered as a substancewhich is within the confines of the tissue cell. When agents which causethe protein cell material to swell are added each individual cellimbibes much water from the natural aqueous juices of the tissue untilso much water has been taken up that the cell wall actually ruptures.Swelling is associated with a great increase in viscosity of the mincedtissue and is directly related to the hydrogen ion concentration oi thetissue. The principle involved can be more easily understood byreference to the action of gelatin.

Normal gelatin will have a hydrogen ion concentration at the isoelectricpoint of about pH 4.7. thereto, the gelatin begins to swell and at a pHof about 3.2 maximum swelling occurs. The solutlon becomes highlyviscose and gels. This point can be defined as the point of "maximumhydrophillation. In other words, at this hydrogen ion concentration thegelatin has absorbed the maximum amount of water whichit can take up.Further additions of acid then cause a decrease in swelling and inviscosity. Similarly, when dilute alkali is added'to gelatin,

the gelatin begins to swell'and reaches a maximum swelling at a pH ofabout 10. This swelling action is, of course, the result of theindividual cells of the cellular gelatin structure imbibing water sothat the cells themselves greatly increase in size. animal tissue andglands behave similarly to the relatively simple gelatin molecule. Asthe hydrogen ion concentration of the animal tissue increases ordecreases from the isoelectric point, the animal tissue cells begin toswell and finally rupture. It is this principle of protein swelling thatI use in my invention except that the bulk of the water taken up by thetissue cells is derived from the normal tissue juices. At the completionof the swelling, the cells rupture, the hor- When diluted hydrochloricacid is added The complex protein cells of mones within the cells areliberated, and these hormones become apart of the colloidal systemitself. In other words, they havevbeen transferred from within thetissue cell to the colloidal system composed of ruptured, hydrated celltissue and tissue juice. Thereafter all I need to do is add to thecolloid system a substance such as sodium chloride or other inorganicsalt to break the system down, whereupon the proteins precipitate butthe hormones remain in the supernatant tissue juice.

In broad aspects, my process includes the steps of treating mincedanimal tissue or glandular material containing its normal content ofanimal tissue juice with small amounts of either an acid or an alkaliuntil the mixture becomes quite stiff, which indicates maximum swelling,and then add to the mixture a salt, or other ionizable :material whichwill cause swollen proteins to dehydrate and precipitate. Thisdehydration is really the result of changing the hydrogen ion fluid aswater.

This principle of inducing swelling in animal tissues and glands torelease the intracellular hormones can, as stated, be used for theseparation of all of the common hormones as well as enzymes. .Forexample, it can be used for the extraction of epinephrin from-mincedadrenalin glands, hormones from the pituitary gland, the thyroid gland,pancreas, the recovery of sex hormones etc. Whether the animal tissue orgland is to be swollen by addition of an acid or an alkali will dependupon the type of hormone to be extracted. When isolating epinephrin,thyroxine and sex hormones it is better to swell the minced material bythe addition of dilute alkali thereto. Most other hormones areunaflected by the dilute acid used to induce swelling by increasedhydrogen ion concentration.

I shall now give a detailed example of the application of my inventionto the recovery of physiologically active materials from hog livers butit will be understood by those skilled in the art that similar processsteps can be applied to the various other phsiologically activematerials enumerated above. The process of the present invention is ofgeneral application in this art and it would serve no useful purpose todescribe in detail the extraction of physiologically active materialsfrom all of the various tissues and glands containing the same.

Fresh hogslivers are finely divided and minced to as near the colloidalstate as possible with ordinary grinding machines. After this has beendone, tissue cells can be visualized as being in suspension in thenatural tissue juice of the liv-' that there is no possibility ofdestroying heat- "able and precipitates.

labile compounds. In tact, chilling is advantageously employed topositively prevent any tendency for enzyme action or deterioration tooccur.

To 100 pounds of this pulped mixture I then add a sulfuric acid solutioncomposed oi pounds of water and about 1130 cubic centimeters ofconcentrated sulfuric acid. is, of course, an extremely dilute solutionof sulfuric acid but the amount of the solution, a little over one-tenthof the weight of the minced liver, is far less than would ever be usefulin extraction processes as hitherto employed. Advantageouely thesulfuric acid solution is cooled to about the temperature or the liverand then added very slowly with vigorous agitation. The mixture stlfienspromptly, thus indicating that the increase in hydrogen ionconcentration of the protein cells in the tissue has caused the materialto swell. Aiter all the acid has been added, the mixture is Quite thlclrand jelly-like and at this calm the colloidal system comprises swollenpro teln cells which have ruptured, animal tissue juices, andphysiologically active materials free from the confines oi the cells. Ithen add 2&3 pounds of sodium chloride to the mixture. The sodiumchloride dissolves in the aqueous phase and causes the gel to hreal:down. The protein mat lal composing the cells is rendered insol-Moreaccurately speakthe sodium chloride causes an immediate decrease inthe hydrogen ion concentration of protein with an immediate decrease inthe swelling so that the protein approaches its isoelectric point,becomes insoluble, and precipitates. The physiologically active materialis in th clear amber colored supernatant liquor. Advantageously, themixture prior to filtration is allowed to settle over night at roomtemperature, about t C. in order to allow ample time for the proteins togranulate.

The next clay the mixture is filtered and the filtrate recovered forfurther treatment. The filtrate may contain some nucleic acids orfragments thereof and also some acid meta-proteins. in order to removethese, I advantageously neutralize the filtrate with caustic sodasolution until the pH is increased to about 5 or 6. At this point awhite curd appears which can be readily filtered off. The filtrate isconcentrated in vacuo at a low temperature to dryness. The temper= atureneed not exceed normal room temperature during the evaporation. Thedried material thus obtained is composed of sodium chloride and thephysiologically active material together with very small amounts ofimpurities such as, sugars and proteins showing the loiuret reaction.The dry material is then ground fine and extracted with absolute methylalcohol or other solvent, filtered, and the filtrate concentrated to aheavy paste in vacuo. This extraction freesthe material of its saltcontent. advantageously the paste is again taken up in a very smallamount of methyl alcohol, just enough to give a filterable mass, andagain filtered. Methyl alcohol is used in the manner described in orderto free the physiologically active material of salt and the lower formsof proteins within the peptone range. The

filtrate last mentioned is next mixed with three volumes of dry acetone,which precipitates the physiologically active material, thephysiological active material is filtered off, and spread thin onenameled pans upon .which it is dried in vacuum. The final product isfree of biuret producing proteins and does not give a Fehlings test forsugar. The hormone can be dissolved in water and used for intramuscularinjections or for oral administrations.

sometimes it is advantageous to freeze the liver ure while it is at itspoint of maximum hydration after the addition of sulfuric acid. :1" thefreezing, water within the cellwalls. or within those cells which havenot ruptured, crystallizes and thus helps to break up the cells. Thisfreezing step is, however, not usually essential and is simply anadjunct in my pro After the freezing operation the material is thawedand salt added as described above.

From the foregoing description it will he anoarent that the essence offmy invention resides in the alumni tissue or glands to swell whichresults in rupturing the cells and release of hormones. Any of thecommon neral acids, or organic acids are water soluble, and

any of the common aliralis can he used to so .cnange the hydrogen ionconcentration I that swelling realized. Sulfuric acid is one of hastacids to use since it not only lowers the pill value "out it alsoprecipitates histories nrota ees. The hydrochlorlolm acids of thesesuostances are soluble whereas the l'lydrosuliates are not.

Those skilled in the art may readily determine the precise quantity ofacid or alirali necessary to maximium swelling by simply treating theminced glands or tissue with a dilute solution, in small amount, oreither an acid or an alkali until the goolnt of maximum viscosity orgelling occurs. This may he determined readily lay visual inspection.

There are different inorganic salts which can he used to hrealr thecolloid system after maximum swelling has been achieved. Sodium chlorideis one oi the best but I do not wish to he limited thereto. Sodiumsulfate, sodi phosphate, calcium chloride, and other salts can he used.

The amount thereof to he used may, of course, vary. Here again visualinspection will readily show when enough salt has been added. Salt isadded until the gel has been completely broken down and the supernatantliquid is easily filtered from the precipitated protein. After thecolloid system has been broken by the addition of a salt thereto anadditional quantity of water,

amounting to not more than about it percent of the original weight ofthe liver can he added but to no particular advantage. The precipitatedprotein material formed after the addition of the salt can, of course,be pressed to remove the last traces of available juice.

Those process steps following the recovery of the asueous tissue juicecontaining the hormone but freed from the cell proteins will be obviousto those skilled in the art. They simply involve evaporation of theanimal juice, separation of salt from the dried product, and removal oiany remaining slight traces of protein decomposition products. Itherefore do not intend to be limited to any particular method ofworking up the solution of hormones in the animal tissue juice.

For purposes of extracting hormones from the duodenum the procedure issubstantially the same. The duodenum is finely minced, 9. small amountof dilute acid added thereto until the material swells to a geily-likemass, salt is then added to break down the gel and precipitate theruptured protein cells which are filtered ofl and the aqueous filtratecompound of the tissue juice, hormone, and salt is then worked up forthe isolation of the hormone. The simplest way to do this is thatdescribed above in connection with the treatment of hog livers.

In the isolation of thyroxine, the thyroid gland is minced to an aqueouspulp, dilute caustic alkali added until a point of maximum swelling,usually at a pH of about II, is reached, salt then added and the aqueousmixture freed -of the precipitated proteins and worked up for theremoval of salt therefrom and precipitation of any residual proteins.

In the appended claims I use the language animal tissue to denotehormone-containing animal tissue and glands. Likewise I use the wordabsorption" to designate the action of the proteins in taking up water.Absorption, used in this way, is intended to be the same as hydration"or occlusion", words which are also used in the art to denote theswelling of proteins on taking up water.

Having thus described my invention, what I claim is:

l. The process of isolating intracellular physiologically activesubstances from animal tissue containing the same which comprises finely.mincing the animal tissue to form a suspension of protein tissue cellsin a liquid menstruum composed substantially of the natural tissue juiceitself without the addition of substantial quantities of aqueous fluidsthereto, changing the hydrogen ion concentration of the mixture of cellsand juice to cause the cells to swell and rupture by absorption oftheliquid menstruum composed substantially of the tissue juice and withoutthe addition of substantial amounts of water so that the mass assumes aplastic and gelatinous state composed of swollen ruptured protein cellmaterial and a liquid menstruum composed substantially of the tissuejuice itself and active substances released from the confines of thecells, and then separating the protein cell material from said liquidmenstruum.

2. The process of isolating intracellular physiologically activesubstances from animal tissue containing the same which comprises finelymincing the animal tissue to form a suspension of protein tissue cellsin a liquid menstruum composed substantially of the natural tissue juiceitself without the addition of substantial quantities of aqueous fluidsthereto, changing the hydrogen ion concentration of the mixture of cellsand juice to cause the cells to swell and rupture by absorption of theliquid menstruum composed substantially of the tissue juice and withoutthe addition of substantial amounts of water so that the mass assumes aplastic and gelatinous state composed of swollen ruptured protein cellmaterial and a liquid menstruum composed substantially of the tissuejuice itself and active substances released from the confines of thecells, precipitating protein cell material from said liquid menstruum,separating the precipitated cell materlal,and recovering thephysiologically active substances from the liquid menstruum.

3. The process as in claim 1 wherein the protein cells are caused toswell and rupture by increasing the hydrogen ion concentration of themixture of cells and natural tissue juice.

4. The process as in claim 2 wherein the protein cells are caused toswell and rupture by increasing the hydrogen ion concentration of themixture of cells and natural tissue juice.

5. The process of obtaining physiologically active substances fromanimal livers which comprises finely mixing the livers to form asuspension of tissue cells in a liquid menstruum composed substantiallyof the natural tissue juice without the addition of substantialquantities of aqueous fluids thereto, adding a small amount of dilutemineral acid to the minced liver material, much less than the quantityof livers, to cause the tissue cells to swell and rupture by absorptionof aqueous fluids present composed substantially of the natural tissuejuice and thus release the physiologically active substances, the

substances, and recovering the physiologically ac-' tive substances fromthe liquid menstruum.

6. The process as in claim 5 wherein the mineral acid is sulfuric acid.

HAVARD L. KEIL.

